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K Ivory

Publications and source records attributed to K Ivory.

At least 19 recordsLinked to original sources
PubMed2001

The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein-Barr virus infection.

Acute infectious mononucleosis (AIM) induced by Epstein-Barr virus (EBV) infection is characterized by extensive expansion of antigen-specific CD8+ T cells. One potential consequence of this considerable proliferative activity is telomere shortening, which predisposes the EBV-specific cells to replicative senescence. To investigate this, a method was developed that enables the simultaneous identification of EBV specificity of the CD8+ T cells, using major histocompatibility complex (MHC) class I/peptide complexes, together with telomere length, which is determined by fluorescence in situ hybridization. Despite the considerable expansion, CD8+ EBV-specific T cells in patients with AIM maintain their telomere length relative to CD8+ T cells in normal individuals and relative to CD4+ T cells within the patients themselves and this is associated with the induction of the enzyme telomerase. In 4 patients who were studied up to 12 months after resolution of AIM, telomere lengths of EBV-specific CD8+ T cells were unchanged in 3 but shortened in one individual, who was studied only 5 months after initial onset of infection. Substantial telomere shortening in EBV-specific CD8+ T cells was observed in 3 patients who were studied between 15 months and 14 years after recovery from AIM. Thus, although telomerase activation may preserve the replicative potential of EBV-specific cells in AIM and after initial stages of disease resolution, the capacity of these cells to up-regulate this enzyme after restimulation by the persisting virus may dictate the extent of telomere maintenance in the memory CD8+ T-cell pool over time.

Acute Disease↗
PubMed2000

Fluticasone propionate-induced regulation of the balance within macrophage subpopulations.

In asthma, treatment with inhaled corticosteroids reduces chronic peribronchial inflammation and restores the balance within macrophage subpopulations. This study investigates whether corticosteroids can regulate monocyte differentiation in vitro and thereby influence the balance of functionally distinct macrophages. Graded doses of fluticasone propionate (FP) were added to cultures of normal peripheral blood monocytes in the presence or absence of IL-4. Cells were harvested after 7 days' culture. Double immunofluorescence studies were performed on cytospins of differentiated macrophages using the MoAbs RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes. Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring tumour necrosis factor-alpha (TNF-alpha) production. FP reduced the number of mature cells with a D1+ antigen-presenting phenotype and up-regulated the development of cells with the D1/D7+ and D7+ phenotypes. Functionally, this was associated with reduced stimulation of T cell proliferation in a mixed leucocyte reaction (MLR). Fluticasone also reversed the increase in both D1+ expression and TNF-alpha production induced by IL-4. The effect of FP persisted for 24 h after removal of FP from the culture medium. These results suggest that FP treatment of asthmatics may have a direct beneficial effect by normalizing the macrophage subset imbalance that contributes to the chronic peribronchial inflammation present in this condition.

Adrenal Cortex Hormones↗
PubMed1996

Leukemia-associated changes identified by quantitative flow cytometry. IV. CD34 overexpression in acute myelogenous leukemia M2 with t(8;21).

During the immunodiagnosis of 517 cases of acute myelogenous leukemia (AML) entered into the Medical Research Council (MRC) AML 10 trials, we have observed the CD34 precursor cell antigen more frequently in AML of M2 morphology, especially in the 84% of cases with the t(8;21) chromosomal translocation, than in any other French-American-British classification group. CD34 expression was then quantified (using QIFI and Quantum Simply Cellular beads [Flow Cytometry Standards, Research Triangle Park, NC] and CD34+ standard cells). When CD34 antibody-binding capacity (ABC) of normal bone marrow (BM) precursors and leukemic blasts was compared, it was shown that AML M2 cases with t(8;21) not only had the highest percentages of CD34+ blasts, but in > 80% of CD34+ cases the individual blasts expressed higher than normal levels of CD34 antigen (> 60 x 10(3) ABC per cell). In addition, in 73% of this group CD34 antigen was overexpressed in an asynchronous combination with cytoplasmic myeloperoxidase (MPO). Other signs of asynchrony included high CD34 expression with CD15 and/or CD56, as well as aberrant combinations of CD13 with terminal deoxynucleotidyl transferase (TdT) and CD19. These findings demonstrate that asynchrony is identifiable in virtually every case of AML with t(8;21), although it does not always involve the same antigens. M2 cases with t(8;21), mostly CD34+, had a 100% remission rate and 71% 5-year survival rate; other patients with CD34+ or CD34- AML showed 69% and 84% remission rates and 31% and 36% 5-year survival rates, respectively. Consequently, individual markers such as CD34 should be interpreted in relation to other features such as chromosomal changes. These simple methods, which are well suited to quantify the expression of ligands, are a useful contribution to diagnosis: 60% to 65% of M2 cases with t(8;21) are rapidly identified by CD34 overexpression alone. This aberration, together with the other signs of asynchrony seen at presentation, can be used to search for residual leukemia after therapy.

Adolescent↗
PubMed1996

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry.

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20 h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL-2 than the other two cytokines, and that there are more TNF-alpha-positive CD4+ cells than cells with IFN-gamma. For normal CD8+ cells the highest production of cytokine is of IFN-gamma (up to 50% of the cells) especially at longer times (10-20 h) of stimulation. For CD8+ cells, IL-2-positive cells exceed those with TNF-alpha. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20 h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-alpha but significantly raised levels of production of IFN-gamma in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.

Adult↗
PubMed1995

Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia.

The expression of bcl-2 protein that is involved in preventing apoptosis in hemopoietic and other cells was evaluated by quantitative flow cytometry in various subpopulations in the normal fetal bone marrow (FBM) and in different types of acute myelogenous leukemias (AML). In the FBM the highest bcl-2 levels (mean antibody binding capacity 51 x 10(3) molecules/cell) were found in CD34+ intermediate sized blasts and myeloblasts, while a CD34+ subset of CD10+ lymphoblasts had low bcl-2 content (8-10 x 10(3) molecules/cell) and the CD34-, CD10+ lymphoblasts were, as expected from previous studies, bcl-2- (< 5 x 10(3) molecules/cell). Variable levels of bcl-2 (5.1-222 x 10(3)) were found in 43 tested cases of AML. The bcl-2 levels decrease with granulocytic and monocytic differentiation and, accordingly, cases of AML with M1 and M2 features showed significantly higher mean bcl-2 levels than the leukemias with promyelocytic (M3) or myelo-monocytic (M4/M5) features. Nevertheless, in seven cases of AML the bcl-2 levels were higher than seen in the normal FBM cells and none of these patients remain in remission after 2 years. Furthermore, in several AML cases intraclonal heterogeneity was observed. The undifferentiated smaller blasts with Class-IIdim display had higher bcl-2 content than the more differentiated larger blasts with more granular side scatter and Class-bright expression. In the same subsets of AML blasts the proliferative S-G2-M fractions showed a reciprocal correlation to bcl-2 content. Thus the higher bcl-2 levels may give a survival advantage and confer some degree of drug resistance to the least differentiated blast populations. The multi-parameter analysis described in this paper, including a combined bcl-2 and cytokinetic analysis of phenotypically defined subgroups of AML blasts, may detect early population shifts during relapse and also guide combination drug therapy.

Antigens, CD↗
PubMed1994

Leukemia-associated changes identified by quantitative flow cytometry: I. CD10 expression.

We have compared CD10 antigen expression in normal fetal bone marrow with that of B-linage acute lymphoblastic leukemia (ALL). Both quantitative indirect immunofluoresence (QIFI) and direct immunofluorescence (IF) tests with Quantum beads were used to convert median fluorescence intensity (MFI) values into numbers of antigen molecules expressed per cell (AgE). Lymphoid precursors in the fetal marrow and liver expressed 3-12.5 x 10(3) CD10 molecules/cell with an upper limit of 5 x 10(4)/cell (MaxAgE). The median CD10 AgE in the different cases of acute B-lineage ALL were variable and ranged from undetectable to very high values (> 1.8 x 10(5). In 24 of the 72 cases (33%) tested with QIFI the median CD10 AgE was above the highest values seen in normal samples (> 5 x 10(4)/cell). An additional 23.6% of cases had higher median values than the normal median CD10 AgE. Next, CD10 antigen was quantitated in 78 cases during the routine multiparameter analysis of B-lineage leukemia using CD10/class II/CD34 3-color IF test or CD10/TdT 2-color IF test. The aberrant overexpression was confirmed in 43.6% of ALL cases. The CD10bright display suggested ALL diagnosis even when few cells were available for study, e.g., in early relapse and in ALL masquerading as aplastic anemia. The levels of CD10 expression were maintained in relapse. In addition, different CD10 levels were associated with the various chromosomal alterations: high CD10 levels (> 3 x 10(4)/cell) with hyperdiploidy, low CD10 levels (1.8-4 x 10(3)/cell) with the t(1;19) and undetectable levels (< 1.2 x 10(3)/cell) with the t(4;11) translocations.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed1994

Leukemia-associated changes identified by quantitative flow cytometry. III. B-cell gating in CD37/kappa/lambda clonality test.

Normal and malignant B-lymphoid cells were studied for CD37 antigen expression with three-color immunofluorescence (IF) in combination with kappa/lambda light chain staining, and by quantitative immunofluorescence utilizing the QIFI test. Peripheral B cells brightly expressed CD37 antigen (median 80-114 x 10(3) molecules/cell). Moderate to high levels (> 20 x 10(3)/cell) of CD37 expression were detected in 364 in 366 cases of peripheral B-cell disorders including all cases of B-ALL, B-cell lymphomas and B-CLL as well as eight of ten cases of PLL. By contrast, slg- B-cell precursors and other cell types in normal bone marrow (BM) were CD37-/CD37dull (< 10 x 10(3) molecules/cell). The negativity for CD37 or only CD37dull expression was confirmed in 180 of 182 cases of precursor B-ALL and 196 cases of non-B malignancies. Among the CD37 cluster, the RFB7 antibody of IgM class showed the weakest binding to non-B cells. In 64 normal samples of blood and BM the CD37+ gated cells showed normal kappa/lambda ratios as expected, while in 100 cases of B malignancy striking changes such as kappa/lambda monoclonality (79%) and aberrant slg- or sigdull expression (21%) were seen among the gated CD37+ B cells. The CD37/kappa/lambda test identified as few as 0.5% kappa+ or lambda- monoclonal B cells admixed to normal BM: circulating B-lymphoma cells were seen in nine patients with morphologically normal blood count. The discrimination of the Kolmogorov-Smirnov (KS) test for kappa/lambda excess was also improved by CD37+ B gating. Thus CD37+ B-cell gating and kappa/lambda analysis is a simple and sensitive routine test, e.g. when combined with autogating on a Cytoron-Absolute cytometer, for identifying malignant B cells in minimally involved BM and blood.

Antibodies, Monoclonal↗
PubMed1991

Human CD4+CD45R0+ and CD4+CD45RA+ T cells synergize in response to alloantigens.

Alloantigens, unlike recall antigens, activate both CD45RA+ (naive) and CD45R0+ (memory) CD4+ cells to the same extent. These T cell subsets may therefore interact with each other in response to alloantigens on transplanted grafts. We have investigated if the ability of activated CD4+CD45RA+ and CD4+CD45R0+ T cells to produce and respond to interleukin 2 (IL2) and IL4 may be involved in this interaction. After activation, both subsets up-regulate their IL2 receptor (IL2R) and IL4R expression, yet IL4 substantially enhanced the proliferation of the CD4+CD45RA+ but not of the CD4+CD45R0+ T cell subset, while IL2 increased the proliferation of CD4+CD45R0+ but not of the CD4+CD45RA+ T cells. Significantly, the CD4+CD45RA+ T cells synthesized two- to threefold more mRNA for IL2 than the CD4+CD45R0+ subset, while the CD4+CD45R0+ T cells synthesized mRNA for IL4 and interferon-gamma exclusively. The addition of IL2 to alloactivated CD4+CD45R0+ T cells further up-regulated their production of all three lymphokine mRNA; in contrast, IL4 induced an increase in mRNA for IL2 in only the alloactivated CD4+CD45RA+ subset. The reciprocity in the ability of both these CD4+ T cells to synthesize and respond to IL2 and IL4 may provide a rationale for the regulation of lymphokine interactions in vivo. Furthermore, the synergy between these subsets in response to alloantigens, which was directly quantitated by co-culturing CD4+CD45RA+ and CD4+CD45R0+ cells together prior to activation, may potentiate the alloreactivity against transplanted grafts in vivo.

Antigens, CD↗
PubMed1990

Inhibition of alloresponsive naive and memory T cells by CD7 and CD25 antibodies and by cyclosporine.

Human naive and memory T cells can be isolated from each other by their CD45RA and CD45RO expression, respectively. This enables the assessment of their differential sensitivities to immunosuppressive agents for the first time. We have investigated the ability of cyclosporine or CD7 and CD25 antibodies to selectively block alloantigen stimulated naive and memory T cells in vitro. CD7 antibodies blocked the proliferation of naive (P less than 0.025) but not memory T cells in a primary MLR. CD25 antibody inhibited both naive and memory subsets but a significantly greater effect was found on the memory T cells (P less than 0.005). The constitutive CD7 and CD25 antigen expression on resting naive or memory T cells was related to the inhibitory activities of these antibodies on both subsets. Accordingly, naive T cells expressed more CD7 antigen than memory cells while memory T cells displayed low levels of CD25 antigen that was absent from naive populations before activation. Cyclosporine, like CD25 antibody, inhibited both subsets in a primary MLR but had a greater effect on memory cells (P less than 0.02). Memory T cells, therefore, are more dependent than naive cells on IL-2 for proliferation. There was great individual variation in the ability of CsA to block the MLR. The simultaneous addition of CD25 or CD7 antibody together with CsA, however, enhanced the MLR inhibition as the effects of all three were additive. This suggested interference by these agents at different points during T cell activation. Thus, in CsA sensitive individuals, one-tenth of the optimal CsA concentration together with CD25 antibody maintained maximum immunosuppression in vitro. These results demonstrate the possibility of using CD7 and CD25 antibodies for selective inhibition of naive or memory T cells and also the possibility of augmenting the inhibition of and reducing the CsA concentration required for clinically effective immunosuppression.

Antibodies, Monoclonal↗
PubMed1986

Graft rejection following HLA matched T-lymphocyte depleted bone marrow transplantation.

Bone marrow graft rejection following HLA-matched bone marrow transplantation (BMT) for leukaemia has been a rare problem. However, with the introduction of T-lymphocyte depleted BMT, graft rejection is recognized as a new complication. At the Royal Free Hospital (RFH) in London T-depletion is achieved using two monoclonal antibodies with complement mediated lysis. The methodology was extended to other centres and in total 56 patients have received T-depleted, HLA matched BMT. Twelve of 56 patients have had graft rejection. At the RFH three of 41 (7%) patients have had rejection whereas at collaborating centres nine of 15 (60%) patients have had rejection. We have investigated these rejections in order to identify factor(s) responsible. Rejection was not restricted by patient or donor characteristics, nor disease status. Patient management, chemotherapy conditioning, efficiency of T-depletion, graft versus host disease (GvHD), and infection post BMT, were not consistently implicated. The major difference between the RFH and all other centres was in the radiotherapy (RT) conditioning: The RFH prescribed a single fraction of 7.5 Gy total body irradiation (TBI) whilst collaborating centres gave 10 or 12 Gy fractionated TBI. We conclude that the different incidence of rejection (7% v. 60%) relates primarily to the RT conditioning although the mechanisms(s) of rejection remain unknown. We conclude that where T-depleted BMT is used, compensation by more intensive RT conditioning is required in order to avert graft rejection.

Adolescent↗
PubMed1986

Standardization of T-cell depletion in HLA matched bone marrow transplantation.

The IBM 2991 Blood Cell Processor has been used to isolate a mononuclear cell (MNC) fraction from the marrow of 31 allogeneic donors. The MNC fraction was then incubated with a combination of two murine monoclonal antibodies MBG6 (CD6) and RFT8 (CD8) followed by two rounds of treatment with rabbit complement resulting in a marrow inoculum significantly reduced in the number of T-lymphocytes. We report here new specifications for the use of Ficoll-Metrizoate, the method used to calculate T-lymphocyte depletion and the details of our attempts to improve T-depletion. Following marrow transplantation with this T-depleted fraction, 29 patients are evaluable for engraftment, one patient failed to engraft and one died too early for evaluation. Twenty-two had no acute graft-versus-host disease (aGvHD), at a minimum of 60 d, six had grade I acute GvHD and one grade III. No correlation was found between the absolute number of MNC infused and time to engraftment, nor any relationship between the number of residual viable T-lymphocytes in the infused marrow and the incidence of GvHD, but the patient with the most severe aGvHD also had the highest number of T-lymphocytes infused.

Antibodies, Monoclonal↗
PubMed1986

T lymphocyte regeneration after transplantation of T cell depleted allogeneic bone marrow.

The regeneration of T cell subsets was studied with double immunofluorescence marker methods in 37 patients who received HLA matched T lymphocyte depleted bone marrow transplants (BMT) as part of the treatment for their haematological disease. A cocktail of anti-pan-T (CD6: MBG6) and anti-suppressor/cytotoxic-T cell (CD8: RFT8) monoclonal antibodies was used with rabbit serum as a source of cytolytic complement to achieve selective T cell lysis. The T8+ cells reached low normal values around 60 days post-transplant and remained within the normal range throughout the study (greater than 150 days). This observation is in contrast to our previously published results in patients who, after receiving BMT without efficient T cell depletion, had increased numbers of circulation T8+ cells from 60 days post-transplant. In the present study Leu-7+, RFT8- cells reached normal values rapidly but the reconstitution of T4+ lymphocytes was slow: low normal levels were reached only around day 150 following BMT. The degree of graft-versus-host disease (GVHD) seemed to be related to the number of residual T cells infused: two of the three patients who received the highest numbers of T cells developed Grade II III; otherwise GVHD was minimal. Among the clinical parameters studied cytomegalovirus (CMV) immune status moderately influenced reconstitution: at 55-90 days post-transplant T8+ cells were present at the upper normal levels in seven out of 15 patients receiving BMT from CMV seronegative donors, but in none of the 16 individuals receiving BMT from seropositive donors. CMV related complications were relatively uncommon. Thus the most significant factor in preventing 'T8+ cell overshoot' and T cell imbalance during regeneration appears to be the depletion of T (including T8+) lymphocytes from marrow. The differences of T8+ cell reconstitution in this and previous studies may reflect a different regeneration pattern from T cell precursors as opposed to inoculated mature T cells.

Adolescent↗
PubMed1986

Effect of cyclosporine on proteinuria in chronic serum sickness in rats.

A model of chronic serum sickness (CSS) was established in Wistar rats. Rats were given 2 mg of bovine serum albumin (BSA) i.p. five days/week. A group of six male rats were bred which developed albuminuria after three weeks. Rat albumin was measured by radial immuno-diffusion. Albuminuria increased gradually over the first 80 days. On days 87 and 88 rats were given 25 mg/kg methylprednisolone (MP) i.p. Albuminuria increased from 1.1 mg/ml to 1.7 mg/ml. After proteinuria had returned to pre-treatment values, MP was given again with the same result. From day 115 rats were given 25 mg/kg of CYA i.p. for three consecutive days. Albuminuria fell from 0.85 mg/ml to 0.28 mg/ml, and for the first time rats developed microscopic haematuria. After five days proteinuria returned to former levels but then rose to higher levels. On day 146 rats were given CYA and albuminuria fell from 1.7 to 0.45 mg/ml. On day 150 the renal histology was examined by light microscopy and the glomeruli showed no significant abnormalities.

Animals↗
PubMed1985

An immunohistological approach to persistent lymphadenopathy and its relevance to AIDS.

Recent evidence has shown that not only AIDS but also the majority of 'unexplained' persistent, generalized lymphadenopathy (PGL) are related to HTLV-III/LAV infections. The early detection how these changes may proceed to AIDS then become a prime interest. Eleven patients with PGL (10 homosexual males and one heterosexual haemophiliac) have been studied by immunohistology using monoclonal antibodies to dendritic reticulum cells of the germinal centre, T and B lymphocyte subsets, plasma cells and factor VIII, as an endothelial marker. In six cases only follicular and paracortical hyperplasia was detected, while in five other cases destruction of the dendritic reticulum cell network was seen with this sensitive method. This early destruction may explain the release of activated B cells into the circulation and prove to be an ominous prognostic sign, as it appears to correlate with 'prodromal' symptoms. In four out of 11 cases the depletion of T4+ cells in the paracortex was not as severe as in the blood, indicating that T4+ cells may preferentially settle in tissues at the time of T4 lymphopenia. In addition, germinal centres contained an additional patchy infiltration of T8+ cells. A patient with Kaposi's sarcoma did not show germinal centre destruction but did reveal extensive plasma cell infiltrates. Immunohistology may contribute to the definition of prognosis and analysis of disease progression in patients with PGL.

Acquired Immunodeficiency Syndrome↗
PubMed1984

The role of monoclonal antibodies in the prevention of graft versus host disease.

We have previously reported that the elimination of T-lymphocytes (greater than 99%) from the donor bone marrow prevents significant graft versus host disease (GvHD). This has been confirmed by other centres. Many of these groups have applied monoclonal antibodies with cytolytic rabbit complement. In some of these studies mostly patients with fully matched sibling donors have been studied and no further immunosuppression has been given during the regeneration period. The experience here shows that the haemopoietic regeneration (to recover a total leucocyte count of 1.0 X 10(9)/l) has only been minimally delayed (mean 25 days) when compared to patients receiving standard methotrexate (Mtx) GvHD prophylaxis (22 days). The incidence of cytomegalovirus (CMV) infections (14%; none fatal) was lower than that in our previous 54 patients (43% CMV infections, 13% fatal). Furthermore, no fatal pneumonitis was seen. The regeneration of T-cells in our patients has shown normal values of T8-positive cells in the circulation from 45 to 50 days onwards, as opposed to the previous groups of patients whose T8-positive cells regenerated to 3-4 times higher than normal values. These observations indicate that T-cell depletion with monoclonal antibodies is an effective method for preventing GvHD, but further studies are necessary to investigate the cause(s) of a new occasional complication, the rejection of the newly established bone marrow.

Adolescent↗